Polymerization of biosynthetic intermediates into bacterial peptidoglycan is essential for growth and survival of bacterial cells, and thus represents a novel target for discovery and design of new antibacterial compounds for clinical use. Bacterial cell wall synthesis requires a functional transglycosylation system in order to polymerize cell wall intermediates into peptidoglycan. The biochemical pathway of peptidoglycan synthesis in bacteria initiates in the cytoplasm with the synthesis of two key nucleotide-linked intermediates, UDP-N-acetyl-muramyl-peptide (“UDP-MurNAc-peptide”, or “MurNAc”) and UDP-N-acetylglucosamine (“UDP-GlcNAc”, or “GlcNAc”). UDP-MurNAc-peptide (which may be in the form of UDP-MurNAc-pentapeptide or UDP-MurNAc-tetrapeptide) is transferred via the MraY protein to a C55 undecaprenol phosphate carrier lipid, thereby generating MurNAc-pentapeptide-pyrophosphoryl-undecaprenol (“lipid I”). N-acetylglucosamine is then transferred to lipid I by the MurG enzyme, generating N-acetylglucosamine-β-1,4-MurNAc-pentapeptide-pyrophosphoryl-undecaprenol (“lipid II”). Lipid II is then polymerized into peptidoglycan by specific transglycosylases.
Presently available methods for assaying transglycosylase activity rely on exogenously supplied substrate, and thus require laborious purification procedures that yield only small amounts of substrate for use in measuring enzymatic activity. In addition, the chemical state of the isolated substrate is difficult to monitor and control, leading to variable activity for the isolated substrate. Thus the ability to detect and characterize inhibitors of the transglycosylation reaction by screening large numbers of compounds for chemical and natural product inventories is not feasible. Furthermore, the lack of available methods for assaying transglycosylase activity directly renders it difficult to distinguish among inhibitors which exert their inhibitory effects at different stages of the peptidoglycan synthesis pathway.
The known transglycosylases are either monofunctional or bifunctional enzymes. Bifunctional enzymes comprise a domain for catalyzing the transglycosylation reaction and a domain catalyzing the transpeptidation reaction cross-linking nascent peptidoglycan to the pre-existing peptidoglycan, while monofunctional transglycosylase enzymes lack a transpeptidase domain. The bifunctional class is also referred to as penicillin-binding-proteins (“PBPs”), due to the fact that the transpeptidase domain can bind penicillin. Examples of this class of bifunctional PBPs are PBP1A, 1B, 2, and 3 from E. coli. Matsuhashi, M. 1994, Utilization of lipid linked precursors and the formation of peptidoglycan in the process of cell growth and division: Membrane enzymes involved in the final steps of peptidoglycan synthesis and the mechanism of their regulation, pp. 55-72. In J.-M. Ghuysen and R. Hakenbeck (ed.), Bacterial Cell Wall: New Comprehensive Biochemistry, vol. 27. Elsevier, N.Y. Matsuhashi et al. suggest that the ability to directly measure transglycosylase activity may depend on the form of the lipid II substrate and precise reaction conditions. In addition, they suggest that PBP2 and PBP3 may require the accessory proteins RodA and FtsW, respectively, for maximal activity. PBP1C was recently identified in the complete E. coli genome sequence, and appears to represent a new, bifunctional enzyme. Schiffer, G., M. Templin, and J. Holtje, 1997, Cloning and biochemical characterization of the bifunctional penicillin-binding protein 1 C from Escherichia coli, GenBank: accession no. U88571.
Monofunctional transglycosylases also exist in bacteria. These enzymes lack a transpeptidase domain, but these enzymes have the ability to synthesize uncrosslinked peptidoglycan in vitro. The presence of monofunctional transglycosylases is not unique to E. coli. Nisseria gonorrhoeae, Haemophilus influenzae, and Klebsiella pneumoniae, all appear to contain genes for monofunctional transglycosylases, as do Staphylococcus aureus and Alcaligenes eutrophus. Spratt, B. G., J. Zhou, M. Taylor, and M. J. Merrick, 1996, Monofunctional biosynthetic peptidoglycan transglycosylases, Mol Microbiol. 19(3):639-40; Di Berardino, M., A. Dijkstra, D. Stuber, W. Keck, and M. Gubler, 1996, The monofunctional glycosyltransferase of Escherichia coli is a member of a new class of peptidoglycan-synthesising enzymes. FEBS Lett. 392(2):184-8. In addition, Bacillus megaterium, Staphylcoccus aureus and Micrococcus luteus, and Streptococcus pneumoniae, exhibit monofunctional transglycosylase activity. Taku, A., M. Stuckey, and D. P. Fan, 1982, Purification of the peptidoglycan transglycosylase of Bacillus megaterium, J Biol Chem. 257(9):5018-22; Park, W., and M. Matsuhashi, 1984, Staphylococcus aureus and Micrococcus luteus peptidoglycan transglycosylases that are not penicillin-binding proteins, J Bacteriol. 157(2):538-44; Park, W., H. Seto, R. Hakenbach, and M. Matsuhashi, 1985, Major peptidoglycan transglycosylase activity in Streptococcus pneumoniae that is not a penicillin binding protein, FEMS Microb. Lett. 27:45-48. The role that these enzymes play in cellular peptidoglycan synthesis is not presently known. The essential nature and roles of several high molecular weight PBPs have been investigated by mutational analysis. The most recent study created E. coli mutants lacking all possible combinations of eight PBPs, and confirmed the need for at least PBP1A or 1B for growth. Denome, S. A., P. K. Elf, T. A. Henderson, D. E. Nelson, and K. D. Young, 1999, Escherichia coli mutants lacking all possible combinations of eight penicillin binding proteins: viability, characteristics, and implications for peptidoglycan synthesis, J Bacteriol. 181(13):3981-93. S. pneumoniae contains six identified PBPs, and expression of PBP1A or 2A is required for growth. Paik, J., I. Kern, R. Lurz, and R. Hakenbeck, 1999, Mutational analysis of the streptococcus pneumoniae bimodular class A penicillin-0117 binding proteins [In Process Citation]. J Bacteriol., 181(12):3852-6. The pbpA gene, encoding PBP 1 in Staphylococcus aureus appears to be essential for growth. Wada, A., and H. Watanabe, 1998, Penicillin-binding protein 1 of Staphylococcus aureus is essentialfor growth, J Bacteriol. 180(10):2759-65.
The initial assays developed to measure transglycosylase activity required purification of radiolabeled, native lipid II from bacterial cells. See, e.g., Ishino, F., and M. Matsuhashi, 1981, Peptidoglycan synthetic enzyme activities of highly purified penicillin-binding protein 3 in Escherichia coli: a septum-forming reaction sequence, Biochem Biophys Res Commun., 101(3):905-11; Ishino, F., K. Mitsui, S. Tamaki, and M. Matsuhashi, 1980, Dual enzyme activities of cell wall peptidoglycan synthesis, peptidoglycan transglycosylase and penicillin-sensitive transpeptidase, in purified preparations of Escherichia coli penicillin-binding protein 1A, Biochem Biophys Res Commun. 97(1):287-93; Ishino, F., W. Park, S. Tomioka, S. Tamaki, I. Takase, K. Kunugita, H. Matsuzawa, S. Asoh, T. Ohta, B. G. Spratt, et al., 1986, Peptidoglycan synthetic activities in membranes of Escherichia coli caused by overproduction of penicillin-binding protein 2 and rodA protein, J Biol Chem. 261(15):7024-31; Suzuki, H., Y. van Heijenoort, T. Tamura, J. Mizoguchi, Y. Hirota, and J. van Heijenoort, 1980, In vitro peptidoglycan polymerization catalysed by penicillin binding protein 1b of Escherichia coli K-12, FEBS Lett. 110(2):245-9, Van Heijenoort, Y., M. Derrien, and J. Van Heijenoort, 1978, Polymerization by transglycosylation in the biosynthesis of the peptidoglycan of Escherichia coli K 12 and its inhibition by antibiotics, FEBS Lett. 89(1):141-4; van Heijenoort, Y., and J. van Heijenoort, 1980, Biosynthesis of the peptidoglycan of Escherichia coli K-12: properties of the in vitro polymerization by transglycosylation, FEBS Lett. 110(2):241-4.
This approach has significant limitations: 1) the amount of lipid II in bacterial cells is small, approximately 2000 molecules per cell (see, e.g., van Heijenoort, Y., M. Gomez, M. Derrien, J. Ayala, and J. van Heijenoort, 1992, Membrane intermediates in the peptidoglycan metabolism of Escherichia coli: possible roles of PBP 1b and PBP 3. J Bacteriol. 174(11):3549-57 [published erratum appears in J Bacteriol. 1992 September;174(18):6004]); 2) purification is a lengthy process, with significant loss of material occurring during purification (see, e.g., van Heijenoort, supra and references cited therein); 3) little is known about the structure of purified lipid II and its ability to be used efficiently as a substrate (see, e.g., van Heijenoort, supra; Matsuhashi supra); 4) the lipid II has to be reintroduced into bacterial membranes in order to be a substrate for a transglycosylase; and 5) only a fraction of the lipid II substrate is enzymatically converted to peptidoglycan product. All of these limitations impair the ability to monitor polymerization of lipid II into peptidoglycan by the action of a transglycosylase enzyme, therefore, the use of purified lipid II as a substrate to perform the large number of transglycosylase assays required for identifying inhibitors in chemical or natural product inventories is neither practical nor feasible. In addition, this assay format limits the ability to further characterize the known natural product inhibitors such as moenomycin and analogs thereof, as well as any newly identified inhibitors of transglycosylase function. See, e.g., El-Abadla, N., M. Lampilis, L. Hennig, M. Findeisen, P. Welzel, D. Muller, A. Markus, and J. van Heijenoort, 1999, Meonomycin A: The role of the methyl group in the moenuronamide unit and a general discussion of structure-activity relationships, Tetrahedron, 55:699-722; M. Ge, Z. Chen, H. R. Onishi, J. Kohler, L. L. Silver, R. Kerns, S. Fukuzawa, C. Thompson and D. Kahne, (1999), Vancomycin derivatives that inhibit peptidoglycan biosynthesis without binding D-Ala-D-Ala, Science. 284:507-11.
The most recent attempt to improve on the ability to conduct transglycosylase assays relied on modifications of the methods used in preparation and purification, but only improved the yield of lipid II substrate from 2% to 18%. van Heijenoort, Y., M. Gomez, M. Derrien, J. Ayala, and J. van Heijenoort, 1992, Membrane intermediates in the peptidoglycan metabolism of Escherichia coli: possible roles of PBP 1b and PBP 3, J Bacteriol. 174(11):3549-57 [published erratum appears in J Bacteriol 1992 September;174(18):6004].
Some antibiotics, such as vancomycin, inhibit peptidoglycan synthesis by binding to cell wall intermediates that contain a dipeptide D-ala-D-ala moiety. A. Malabarba, T. I. Nicas and R. C. Thompson, (1997), Structural modifications of glycopeptide antibiotics, Med Res Rev. 17, 69-137; R. Nagarajan, (1993), Structure-activity relationships of vancomycin-type glycopeptide antibiotics, J Antibiot (Tokyo), 46, 1181-95. Unfortunately, the increased clinical use of vancomycin has selected for vancomycin resistant Enterococci, which replace the final D-ala of the dipeptide with a lactate moiety (D-lac), such that D-ala-D-ala is replaced with D-ala-D-lac, leading to a 1000-fold decrease in drug:ligand interaction and MIC. C. T. Walsh, S. L. Fisher, I. S. Park, M. Prahalad and Z. Wu, (1996), Bacterial resistance to vancomycin: five genes and one missing hydrogen bond tell the story, Chem Biol. 3, 21-8; C. T. Walsh, (1993), Vancomycin resistance: decoding the molecular logic, Science. 261, 308-9 [published erratum appears in Science Oct. 8, 1993; 262(5131):164]; R. Leclercq and P. Courvalin, (1997), Resistance to glycopeptides in enterococci, Clin Infect Dis. 24, 545-54; R. Leclercq, (1999), Glycopeptides: mechanism ofaction and of resistance, Presse Med., 28, 720-1; M. Arthur, P. E. Reynolds, F. Depardieu, S. Evers, S. Dutka-Malen, R. Quintiliani, Jr. and P. Courvalin, (1996), Mechanisms of glycopeptide resistance in enterococci, J Infect. 32, 11-6. However, certain natural product and semi-synthetic glycopeptide analogs are known which have potent activity against vancomycin resistant Enterococci. See, e.g., Malabarba, supra; M. Ge, Z. Chen, H. R. Onishi, J. Kohler, L. L. Silver, R. Kems, S. Fukuzawa, C. Thompson and D. Kahne, (1999), Vancomycin derivatives that inhibit peptidoglycan biosynthesis without binding D-Ala-D-Ala, Science. 284, 507-11. These include teicoplanin, eremomycin, chloroeremomycin, and novel vancomycin analogs.
The antibacterial activity of such novel compounds against bacteria that lack the D-ala-D-ala binding site was explained based on two fundamentally different hypotheses. The first evokes a combination of membrane anchoring and drug dimerization for specific glycopeptide analogs that leads to enhancement of the weak interaction with D-ala-D-lac. D. H. Williams, (1996), The glycopeptide story—how to kill the deadly ‘superbugs’, Nat. Prod. Rep. 13, 469-77; N. E. Allen, D. L. LeToumeau and J. N. Hobbs, Jr., (1997), Molecular interactions of a semisynthetic glycopeptide antibiotic with D-alanyl-D-alanine and D-alanyl-D-lactate residues, Antimicrob. Agents Chemother., 41, 66-71.
The second proposes inhibition of the transglycosylases responsible for polymerization of intermediates into peptidoglycan. Ge et al., supra. Until the present invention, there was no straightforward method available for determining which of these hypotheses was correct for particular inhibitors. The present invention's transglycosylase assay provides a method of determining which of these hypotheses is correct, using the UDP-MurNAc-tetrapeptide substrate, which lacks the D-ala-D-ala dipeptide.
Additionally, until the present invention, there was no simple, reproducible assay for monitoring the enzymatic activity of bacterial transglycosylase. Thus the ability to discover and validate inhibitors of transglycosylasc function by assaying large numbers of compounds in chemical or natural product inventories was not feasible. In addition, it was difficult to further characterize the known natural product inhibitors of transglycosylase activity, such as moenomycin, or any other putative transglycosylase inhibitors.
Co-owned U.S. Provisional Patent Application No. 60/097,324, filed Aug. 20, 1998, discloses an assay employing a biotinylated MurNAc-pp substrate, which biotin moiety allows for the capture, separation, and measurement of lipid II and peptidoglycan, and the subsequent testing of potentially inhibitory compounds. However, the disclosed method is still unable to assess which step in the biosynthetic pathway is being inhibited.
U.S. Provisional Patent Application Ser. No. 09/241,862 discloses synthetic analogs of lipid I for use in assays measuring transglycosylase activity, however, the disclosed assays require the synthesis of such analogs. Moreover, the analogs have not been shown to yield lipid II substrate which is active in the transglycosylation step of the peptidoglycan synthesis pathway.
U.S. Provisional Patent Application No. 60/122,966 discloses synthetic analogs of lipid II for use in assays directly measuring transglycosylase activity, however, the disclosed assays require the synthesis of such analogs. Moreover, the analogs have not been shown to act as substrate for the transglycosylation step of the peptidoglycan synthesis pathway.
The art is in need of assay methods for directly measuring transglycosylase activity without the need for cumbersome purification of substrate, or production of synthetic analogs, in order to assess potential inhibitors, identify which stages of the biosynthetic pathway are being inhibited, and ascertain the likelihood that such identified inhibitors would be effective against otherwise resistant bacteria.